Calcium dynamics during fertilization in C. elegans.

Abstract

BACKGROUND:

Of the animals typically used to study fertilization-induced calcium dynamics, none is as accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated by the experimental possibilities inherent in using such a well-established model organism, we have characterized fertilization-induced calcium dynamics in C. elegans.

RESULTS:

Owing to the transparency of the nematode, we have been able to study the calcium signal in C. elegans fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the cytosol of oocytes. In C. elegans, fertilization induces a single calcium transient that is initiated soon after oocyte entry into the spermatheca, the compartment that contains sperm. Therefore, it is likely that the calcium transient is initiated by contact with sperm. This calcium elevation spreads throughout the oocyte, and decays monotonically after which the cytosolic calcium concentration returns to that preceding fertilization. Only this single calcium transient is observed.

CONCLUSION:

Development of a technique to study fertilization induced calcium transients opens several experimental possibilities, e.g., identification of the signaling events intervening sperm binding and calcium elevation, identifying the possible roles of the calcium elevation such as the completion of meiosis, the formation of the eggshell, and the establishing of the embryo's axis of symmetry.

A DIC image of the poserior arm of the gonad. Oocytes are formed by budding from the syncytial gonad, each bud carrying a single nucleus. Oocytes develop in single-file, and oocytes at various stages of development are visible. Oocytes are fertilized upon entrance to the spermatheca. After leaving the spermatheca, embryos develop in the uterus awaiting expulsion through the vulva. We inject dyes into the syncytial gonad. After recovery, the fluorescence pattern of the dyes becomes that of figure .

A portion of the posterior arm of the gonad. Fluorescence of tetramethylrhodamine, dextran 10,000 MW (red image) and Calcium Green-1, dextran 10,000 MW (green image) simultaneously measured in an injected animal. A single z-section constitutes this confocal image. The white arrow indicates the proximal oocyte, i.e., the oocyte nearest the entrance to the spermatheca. Blue arrows indicate developing embryos within the uterus. Pink arrows indicate the syncytial gonad in which pachytene nuclei line the walls of the syncytium. Yellow arrows denote maturing oocytes. The asterisk (*) denotes the spermatheca.

Fluorescence of Calcium Green-1, dextran 10,000 MW in a fertilized oocyte monitored by CCD camera. Images are presented at 3 sec intervals. The oocyte that is fertilized is denoted by the arrow. In this image, the uterus is above the fertilized oocyte, and contains a 2-celled embryo. An immature oocyte is below the fertilized oocyte, and has not yet undergone nuclear envelope breakdown. At t = 6 sec, the mature oocyte begins to enter the spermatheca through the sphincter at its entrance (at this point the oocyte is not yet encased in eggshell and retains flexibility). Between t = 12 sec and t = 15 sec, the leading edge of the oocyte has engulfed a sperm leading to a local ~ 30% increase in fluorescence, i.e., cytosolic [Ca++]. As the oocyte enters the spermatheca, the [Ca++] elevation spreads throughout the cell. At t = 30 sec, the entire oocyte has entered the spermatheca, and the oocyte has become rigid as a result of the formation of eggshell.

Ratiometric imaging of calcium elevation obtained by confocal microscopic measurement of fluorescence of coinjected tetramethylrhodamine, dextran 10,000 MW and Calcium Green-1, dextran 10,000 MW. Single z-sections constitute these images. These ratiometric images were obtained by calculating Δf/f at each pixel (fCaGreen-fRhodamine)/fRhodamine) which is the fractional increase in the fluorescence of the green channel (Calcium Green-1) over the red channel (tetramethylrhodamine); in all experiments, there was no measurable variation in the amplitude of the tetramethylrhodamine fluorescence (data not shown) making it useful for these ratiometric calculations). The inset is a plot of the average Δf/f within the area of the fertilized oocyte. The fertilized oocyte is denoted by the arrow. The empty spermatheca is denoted by the arrowhead (the redness of the spermatheca is an artifact of the ratio calculation; where there is no dye, green background fluorescence exceeds red background fluorescence, the area outside the worm was painted black for presentation purposes). At t = 3 min, the oocyte has completely entered the spermatheca and has been fertilized. The oocyte has entered the uterus at t = 9 min (the actual entry was missed between scan intervals). After ~ 12 min, cytosolic [Ca++] essentially reaches the level prior to fertilization. At t ~ 40 min, the first cell division takes place (data not shown), thus the calcium dynamic imaged here can be considered physiologically normal and not perturbative.